Development of Diagnosis and Treatment of Malignant Diseases Based on the Expression of DNaseγ

RT-PCR检测GnRH及其受体在人卵巢上皮性癌细胞株(HTOA、OV-1063、OVCAR-3)和人子宫内膜癌细胞株(HEC-1、HHUA)中的表达。此外，RT-PCR法还检测了DNA酶γ和caspase3激活的DNA酶在这些细胞系中的表达。原位末端标记法显示，促性腺激素释放激素激动剂布斯瑞林和促性腺激素释放激素拮抗剂西曲瑞克斯均能增加培养的HTOA细胞的凋亡率。两种类似物对裸鼠HTOA细胞株移植瘤的生长均有相同程度的抑制。在培养的HTOA细胞中，两种促性腺激素释放激素类似物均可抑制EGF诱导的EGF受体酪氨酸磷酸化，并阻断细胞周期于G0/G1期。这些发现提示两种促性腺激素释放激素类似物的抗肿瘤作用可能是通过激活DNA酶、抑制EGF诱导的信号通路或使细胞周期停滞于G1期而诱导的。另一方面，在上述5种细胞系中，用本实验室制备的抗DNA酶γ的多克隆抗体和单抗进行Western blotting均未检测到DNA酶γ蛋白在这些细胞系中的表达。在此基础上又产生了一株高度特异的抗DNA酶γ单抗。该抗体已被证明可用于免疫印迹和酶联免疫吸附试验。

RT-PCR revealed the expression of mRNA for GnRH and its receptor in human epithelial ovarian cancer cell lines (HTOA, OV-1063 and OVCAR-3) and human endometrial cancer cell lines (HEC-1 and HHUA). Furthermore, the expression of mRNA of DNaseγ and CAD (caspase 3-activated DNase) was also detected by RT-PCR in these cell lines. The GnRH agonist, buserelin, and the GnRH antagonist, cetrorelix, increased the incidence of apoptosis in cultured HTOA cells as determined by TUNEL staining. The treatment with both analogs suppressed the growth of xenografts of HTOA cell line in nude mice to the same extent. In cultured HTOA cells, the both GnRH analogs suppressed EGF-induced tyrosine phosphorylation of EGF receptor and blocked cell cycle progression in G0/G1 phase. These findings lead us to suggest that the antineoplastic actions of both GnRH analogs might be based on apoptosis induced by the activation of DNase, suppression of EGF-induced signaling pathway or cell cycle arrest at G1. On the other hand, in the five cell lines described above, the expression of DNaseγ protein could not be detected in these cell lines by Western blotting using anti-DNaseγ polyclonal and monoclonal antibodies generated by us previously. Then a remarkably specific anti-DNaseγ monoclonal antibody was newly generated. This antibody has been shown to be useful for Western blotting and ELISA.